Jeffrey Yachnin

Targeting p53 in Vivo: A First-in-Human Study With p53-Targeting Compound APR-246 in Refractory Hematologic Malignancies and Prostate Cancer

PURPOSE APR-246 (PRIMA-1MET) is a novel drug that restores transcriptional activity of unfolded wild-type or mutant p53. The main aims of this first-in-human trial were to determine maximum-tolerated dose (MTD), safety, dose-limiting toxicities (DLTs), and pharmacokinetics (PK) of APR-246. PATIENTS AND METHODS APR-246 was administered as a 2-hour intravenous infusion once per day for 4 consecutive days in 22 patients with hematologic malignancies and prostate cancer. Acute myeloid leukemia (AML; n = 7) and prostate cancer (n = 7) were the most frequent diagnoses. Starting dose was 2 mg/kg with dose escalations up to 90 mg/kg. RESULTS MTD was defined as 60 mg/kg. The drug was well tolerated, and the most common adverse effects were fatigue, dizziness, headache, and confusion. DLTs were increased ALT/AST (n = 1), dizziness, confusion, and sensory disturbances (n = 2). PK showed little interindividual variation and were neither dose nor time dependent; terminal half-life was 4 to 5 hours. Tumor cells showed cell cycle arrest, increased apoptosis, and upregulation of p53 target genes in several patients. Global gene expression analysis revealed changes in genes regulating proliferation and cell death. One patient with AML who had a p53 core domain mutation showed a reduction of blast percentage from 46% to 26% in the bone marrow, and one patient with non-Hodgkins lymphoma with a p53 splice site mutation showed a minor response. CONCLUSION We conclude that APR-246 is safe at predicted therapeutic plasma levels, shows a favorable pharmacokinetic profile, and can induce p53-dependent biologic effects in tumor cells in vivo.

Pharmacokinetics of oxaliplatin in humans.

Oxaliplatin is a novel platinum complex used for the treatment of metastatic colorectal carcinoma. The pharmacokinetics of the free fraction of oxaliplatin in blood were evaluated in 10 patients given 85 mg/m2 of oxaliplatin using an infusion time of 2 h. Blood samples were collected during and after the infusion and immediately placed on ice. The samples were ultrafiltrated centripetally and the concentration of oxaliplatin in the ultrafiltrate was determined by liquid chromatography in combination with postcolumn derivatization. The in vitro degradation rate was determined in blood from the patients taken immediately before drug administration.The maximal blood concentration (Cmax) and terminal half-life (t1/2) were 1.44±0.20 (SD) µg/mL and 14.1 min (range: 10.2–24.5), respectively. The area under the blood concentration time curve (AUC), clearance (CL), and distribution volume (Vss) were (means±SD) 161±22 µg min/mL, 32.1±4.2 L/h/m2, and 0.26±0.06 L/kg, respectively. There was a significant correlation between the clearance of oxaliplatin in the patients and the degradation rate in whole blood (r=0.746; p=0.017).Oxaliplatin has a short elimination half-life, which is in a sharp contrast to previously reported elimination half-lives obtained by analysis of the platinum content in plasma and ultrafiltrate. The correlation between in vivo and in vitro data suggests that the degradation in whole blood plays a role for the elimination of the drug.

The kinetics and cytotoxicity of cisplatin and its monohydrated complex

This paper examines the monohydrated complex of cisplatin (MHC) with respect to kinetics and cytotoxicity. Equilibrium mixtures of cisplatin and hydrated species have been used in previous studies of a similar nature. To our knowledge, this is the first paper examining MHC after isolation and quantification. This was accomplished using liquid chromatography with porous graphitic carbon. MHC and cisplatin were quantified over time in a suspension of the small-cell lung cancer cell line U-1285. Cytotoxicity was evaluated using the fluorescent microculture cytotoxicity assay. MHC was significantly more cytotoxic than cisplatin at the high end of the drug concentrations tested. In culture media with low chloride ion concentrations, the stability of MHC was related to changes in pH. At a pH of between 6.0 and 7.2, MHC was rapidly converted to cisplatin. In culture media with a pH above 7.2, MHC was considerably more stable. These findings might have clinical significance given that MHC circulates in the blood stream of patients receiving cisplatin infusions and that solid tumours often have environments that are extremely acidotic.

Oxaliplatin neurotoxicity – no general ion channel surface-charge effect

BackgroundOxaliplatin is a platinum-based chemotherapeutic drug. Neurotoxicity is the dose-limiting side effect. Previous investigations have reported that acute neurotoxicity could be mediated via voltage-gated ion channels. A possible mechanism for some of the effects is a modification of surface charges around the ion channel, either because of chelation of extracellular Ca2+, or because of binding of a charged biotransformation product of oxaliplatin to the channel. To elucidate the molecular mechanism, we investigated the effects of oxaliplatin and its chloride complex [Pt(dach)oxCl]- on the voltage-gated Shaker K channel expressed in Xenopus oocytes. The recordings were made with the two-electrode and the cut-open oocyte voltage clamp techniques.ConclusionTo our surprise, we did not see any effects on the current amplitudes, on the current time courses, or on the voltage dependence of the Shaker wild-type channel. Oxaliplatin is expected to bind to cysteines. Therefore, we explored if there could be a specific effect on single (E418C) and double-cysteine (R362C/F416C) mutated Shaker channels previously shown to be sensitive to cysteine-specific reagents. Neither of these channels were affected by oxaliplatin. The clear lack of effect on the Shaker K channel suggests that oxaliplatin or its monochloro complex has no general surface-charge effect on the channels, as has been suggested before, but rather a specific effect to the channels previously shown to be affected.

Energy-requiring uptake of prostasomes and PC3 cell-derived exosomes into non-malignant and malignant cells

Epithelial cells lining the prostate acini release, in a regulated manner (exocytosis), nanosized vesicles called prostasomes that belong to the exosome family. Prostate cancer cells have preserved this ability to generate and export exosomes to the extracellular space. We previously demonstrated that human prostasomes have an ATP-forming capacity. In this study, we compared the capacity of extracellular vesicles (EVs) to generate ATP between normal seminal prostasomes and exosomes secreted by PC3 cells (PC3 exosomes), a prostate cancer cell line. Proteomic analyses identified enzymes of the glycolytic chain in both prostasomes and PC3 exosomes, and we found that both of them were capable of generating ATP when supplied with substrates. Notably, the net production of extracellular ATP was low for prostasomes due to a high ATPase activity contrary to an elevated net ATP level for PC3 exosomes because of their low ATPase activity. The uptake of the 2 types of EVs by normal prostate epithelial cells (CRL2221) and prostate cancer cells (PC3) was visualized and measured, demonstrating differential kinetics. Interestingly, this uptake was dependent upon an ongoing glycolytic flux involving extracellular ATP formation by EVs and/or intracellular ATP produced from the recipient cells. We conclude that the internalization of EVs into recipient cells is an energy-requiring process also demanding an active V-ATPase and the capacity of EVs to generate extracellular ATP may play a role in this process.

The In Vitro Stability of Circulating Tumour DNA.

Objective DNA from apoptotic cancer cells, present in the circulation, has the potential to facilitate genomic profiling and disease monitoring. However, only low fractions of total cell-free DNA originates from cancer cells, limiting the applicability of circulating tumour DNA (ctDNA). Optimal sample processing is consequently of uttermost importance. Therefore, we evaluated the in vitro stability of ctDNA. Experimental design Blood was collected in 10 ml EDTA or Streck tubes. Three conditions (EDTA and Streck tubes in room temperature, EDTA tubes at five degrees) and four time points (plasma harvested from blood aliquots of each 10 ml tube in a time series up to 24 h) were investigated. Each condition was evaluated in five metastatic prostate cancer patients. Subsequently, three additional patients were collected enabling investigation of the in vitro stability in EDTA tubes up to 48 h. Methods The in vitro stability of ctDNA was interrogated by low-pass whole genome sequencing which allows for the identification of somatic copy-number alterations (CNAs). In silico simulations demonstrated that non-parametric testing could detect a 1% contamination by white blood cell DNA. Mutational profiling was performed by targeted, in-solution based hybridization capture and subsequent sequencing. The allelic fraction of individual mutations was used as an estimate of the in vitro stability. Results Somatic CNAs were detected in all patients. Surprisingly, the ctDNA levels at zero hours were not significantly different to 24 or 48 hour in vitro incubation in any investigated condition. Subsequently, mutational profiling corroborated the conclusions from the CNA analysis. Conclusions The stability of ctDNA simplifies logistics without the requirement of immediate processing or applying fixatives to prevent white blood cell lysis.

A QTc study of cabazitaxel in patients with advanced solid tumors.

257 Background: Cabazitaxel (Cbz) improves overall survival in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC) after docetaxel failure, compared with mitoxantrone (HR 0.70; 95% CI 0.59-0.83; P < 0.0001). Following Cbz approval for the treatment of mCRPC, this study was undertaken to evaluate any effect of Cbz on the QTc interval. METHODS This prospective, multinational, open-label study ( NCT01087021 ) enrolled pts with advanced solid tumors (without other therapeutic options). Cbz 25 mg/m² IV was administered on Day 1 Q3W. QTc and other ECG intervals were assessed on Day 1 of Cycle 1. Triplicate ECGs were obtained from 12-lead Holter recordings and concomitant serial blood samples were collected for pharmacokinetic (PK) analysis. The primary endpoint was change from baseline in the corrected QTc interval (according to the Fridericia formula QTcF). RESULTS A total of 96 pts were enrolled; 32 pts under the original protocol (6-h Holter) and 64 pts following protocol amendment 1, which extended ECG and PK monitoring (24-h Holter). Median age was 63 yrs (69.8% male), 30.5% and 57.9% of pts were ECOG PS 0 and 1 respectively; 33 pts (34.4%) had prostate cancer. Screening ECG was abnormal but not clinically significant in 39.6% of pts. The majority (n = 65) of pts received ≥ 3 treatment cycles; safety and ECG parameters were evaluated in 95 and 94 pts, respectively. In the 24-h Holter group (n = 63), the maximum least squares (LS) mean change from baseline in QTcF was 4.8 msec (90% CI 2.1-7.5), returning to baseline by 24 h. Similar results were observed in the overall population (n = 94). At Cmax, Cbz concentration had no effect on QTcF change from baseline; the mean (CV%) Cmax (n = 91) and AUC (n = 92) were 276 ng/ml (63%) and 1245 ng.h/ml (82%). The LS mean change from baseline in heart rate increased up to 24 h but remained < 10 beats per minute. The most common Grade 3/4 AEs were neutropenia (27.4%), febrile neutropenia (12.6%), fatigue (12.6%) and dehydration (5.3%). No Grade 3/4 cardiac AEs were reported. Of the 6 deaths reported, 1 (infection) was study drug related. CONCLUSIONS Cbz had no significant effect on QTc interval in pts with advanced tumors. The Cbz safety profile is consistent with previous findings and with other taxane-based therapies.

Cell-free DNA profiling of metastatic prostate cancer reveals microsatellite instability, structural rearrangements and clonal hematopoiesis

Background There are multiple existing and emerging therapeutic avenues for metastatic prostate cancer, with a common denominator, which is the need for predictive biomarkers. Circulating tumor DNA (ctDNA) has the potential to cost-efficiently accelerate precision medicine trials to improve clinical efficacy and diminish costs and toxicity. However, comprehensive ctDNA profiling in metastatic prostate cancer to date has been limited. Methods A combination of targeted- and low-pass whole genome sequencing was performed on plasma cell-free DNA and matched white blood cell germline DNA in 364 blood samples from 217 metastatic prostate cancer patients. Results ctDNA was detected in 85.9% of baseline samples, correlated to line of therapy and was mirrored by circulating tumor cell enumeration of synchronous blood samples. Comprehensive profiling of the androgen receptor (AR) revealed a continuous increase in the fraction of patients with intra-AR structural variation, from 15.4% during first line mCRPC therapy to 45.2% in fourth line, indicating a continuous evolution of AR during the course of the disease. Patients displayed frequent alterations in DNA repair deficiency genes (18.0%). Additionally, the microsatellite instability phenotype was identified in 3.81% of eligible samples (≥0.1 ctDNA fraction). Sequencing of non-repetitive intronic- and exonic regions of PTEN, RB1 and TP53 detected biallelic inactivation in 47.5%, 20.3% and 44.1% of samples with ≥0.2 ctDNA fraction, respectively. Only one patient carried a clonal high-impact variant without a detectable second hit. Intronic high-impact structural variation was twice as common as exonic mutations in PTEN and RB1. Finally, 14.6% of patients presented false positive variants due to clonal hematopoiesis, commonly ignored in commercially available assays. Conclusions ctDNA profiles appear to mirror the genomic landscape of metastatic prostate cancer tissue and may cost-efficiently provide somatic information in clinical trials designed to identify predictive biomarkers. However, intronic sequencing of the interrogated tumor suppressors challenge the ubiquitous focus on coding regions and is vital, together with profiling of synchronous white blood cells, to minimize erroneous assignments which in turn may confound results and impede true associations in clinical trials.